The invention relates to recombinant microorganisms with environmentally limited growth and viability, and more particularly to recombinant microorganisms that may only survive in a host organism for a defined period of time and will not grow outside of the host organism.
Genetically engineered microorganisms have widespread utility and importance. For example, they can be used to produce foreign proteins, and thus can be used industrially for synthesis of products such as interferons, insulin, and growth hormone; they can also be used as antigen delivery systems to produce an immune response. However, it is undesirable for the genetically-engineered microorganism to persist in the environment.
Physical containment was the first means used to control the spread of genetically engineered microorganisms. More sophisticated means were then developed where microorganisms were contained by introducing debilitative mutations that prevent their growth in the absence of specific growth conditions, such as a particular amino acid. U.S. Pat. No. 4,190,495 discloses microorganisms with multiple mutations to prevent growth or genetic transfer outside of the controlled laboratory conditions.
Suicide plasmids have been described for use in biological containment of microorganisms (Molin et al., Annual Review of Microbiology 47:139-166 (1993)). Suicide plasmids generally use differential expression of a specific gene in and out of a controlled environment to prevent survival of the microorganism outside of the controlled environment. The use of suicide plasmids or systems has been limited to the expression of a required gene, or the repression of a lethal gene, under laboratory or other artificial conditions. In the absence of the supplied stimulus, the lethal gene becomes expressed. Either event leads to cell death either directly or due to a severe competitive disadvantage. Most suicide systems depend upon expression of a gene that is actively toxic to the microorganism. A common problem with active suicide systems is the high selective pressure for mutations in the killing gene. This is analyzed, for example, by Knudsen and Karlstrxc3x6m, Applied and Environmental Microbiology 57(1):85-92 (1991), who used a suicide function controlled by LacIq binding to Plac to repress expression of relF, thus preventing the suicide that would be caused by synthesis of the relF gene product. Expression of RelF, and thus display of the suicide phenotype, required the addition of IPTG. This system therefore served as a laboratory model to study induction of host killing within the plasmid-containing host or after transfer of the plasmid to a host lacking the lacI encoded repressor. The efficacy of the system was limited on the one hand by mutations that made killing by the relF gene product ineffective, and on the other hand by low level expression of relF even in the absence of added IPTG such that cells in the culture grew slowly.
Molin et al., Bio/Technology 5:1315-1318 (1987), describes the use of hok, encoding a small peptide that is lethal when expressed in many bacterial species, to prevent survival of a recombinant microorganism when outside of a controlled fermenter environment. The hok gene is homologous to the relF gene. Molin et al. (1987) uses an invertible promoter to control expression of hok. Stochastic inversion of the inactive promoter to the active orientation causes the bacterial population to dwindle due to the death of a predetermined fraction of the cells per unit time. This stochastic cell death is dependent on the relative expression of the fimB and fimE genes which control the flip-flop orientation of the invertible promoter. This system does not lead to a rapid drop in bacterial population density except under ideal laboratory conditions.
Molin and Kjelleberg, AMBIO 22(4):242-245 (1993), and Ramos et al., Bio/Technology 13:35-37 (1995) have hypothesized that microorganisms for release in natural environments might be regulated by suicide genes. Molin and Kjelleberg describe the use of a suicide gene regulated by general or specific starvation. Ramos et al. disclose the use of gef family genes, of which hok and relF are members, and nuclease genes, as suicide genes. Ramos et al. also described killing by these gene products expressed upon IPTG induction of the inducible Plac fused to the gef gene. Ramos et al. describe regulation of the suicide function by loss of a specific nutrient or condition, such as occurs outside of artificially controlled conditions, and by linking the regulatory stimulus to the task of the microorganism. This was accomplished by controlling the rate of inversion of a promoter which in one orientation causes expression of relF, resulting in cell death. This system, of course, only leads to a gradual loss in the viability of the cell population once the cells are in an environment lacking a nutrient. Ramos et al. also suggests the use of biological containment to make live antigen delivery systems feasible. However, Ramos et al. (page 37) points out that the complexity of the human gut precludes the design of control circuits based on specific stimuli, suggesting containment based on differential growth rates in and out of the gut. Ramos et al. does not suggest any regulatory system that could achieve this goal.
Live bacterial vaccines have been described that express a desired antigen and colonize the intestinal tract of animals (Curtiss et al., Curr. Topics Micro. Immun. 146:35-49 (1989); Curtiss, Attenuated Salmonella Strains as Live Vectors for the Expression of Foreign Antigens, in New Generation Vaccines (Woodrow and Levine, eds., Marcel Dekker, New York, 1990) pages 161-188; Schxc3x6del, Infection 20(l):1-8 (1992); Cxc3xa1rdenas and Clements, Clinical Micro. Rev. 5(3):328-342 (1992)). Most work to date has used avirulent Salmonella typhimurium strains synthesizing various foreign antigens for immunization of mice, chickens and pigs. Several avirulent S. typhi vectors have been evaluated in human volunteers (Tacket et al., Infect. Immun. 60:536-541 (1992)) and several phase I clinical trials with recombinant avirulent S. typhi. strains are in progress in the U.S. and Europe.
Although research progress toward expanding and further improving the recombinant avirulent Salmonella antigen delivery strategy has progressed at a reasonable rate, commercial development of recombinant vaccines for the control of infectious diseases of animals or humans has been slow. An important safety advantage of the live attenuated bacterial vaccine vectors as compared to the use of viral vector based vaccines is the ability to treat an immunized patient with oral ciprofloxacin or amoxicillin, should an adverse reaction occur. However, current live bacterial vaccines have the disadvantage that oral administration leads to fecal shedding, with the potential risk that the bacterial vaccine strain will proliferate in nature and infect individuals not selected for immunization. It is known, for example, that fecal coliforms can persist for extended periods under field conditions, with only moderate reductions in numbers (Temple et al., Appl. Environ. Microbiol. 40:794-797 (1980)). There is also concern that these surviving vaccine strains will transmit their cloned genetic information to more robust microorganisms encountered in nature with not always predictable consequences. Although the transmission of most expressed genes to wild-type microbial species would not be harmful, some recombinant vectors expressing genes for sperm-specific antigens or lymphokines could have adverse consequences if widely disseminated. It is therefore desirable to have a biological containment system regulated by the conditions that differ between the target environment and other environments, or which survives only temporarily in the target environment. In the case of microorganism-based delivery of recombinant expression products, it is desirable to have a microorganism that survives inside the animal, but dies outside of the animal, or which lives and survives inside the animal for sufficient time to induce an immune response, expose the animal to an expression product, and/or to deliver a transfer vector for production of an expression product within cells of the animal, prior to the onset of death within the animal. Live attenuated bacterial antigen delivery vectors with inherent biological containment features to preclude survival, proliferation and gene transfer in nature would increase the acceptability and enthusiasm for use of this type of antigen delivery microorganism.
It is therefore an object of the present invention to provide an Environmentally Limited Viability System for use in controlling viability of targeted microorganisms and limiting the undesirable survival of recombinant extrachromosomal genetic information if transferred to other microorganisms.
It is another object of the invention to provide a live recombinant microorganism with environmentally limited viability that can deliver an expression product or vector to a host organism or other environment.
It is another object of the invention to provide a method of delivering an expression product or vector to a host organism or other environment using a live recombinant microorganism with environmentally limited viability.
It is another object of the invention to provide a live recombinant antigen delivery microorganism with environmentally limited viability.
It is a further object of the invention to provide a method of vaccination using a live recombinant antigen delivery microorganism with environmentally limited viability.
Disclosed is an Environmentally Limited Viability System (ELVS) for microorganisms based on differences between permissive and non-permissive environments. Viability of the microorganisms are limited to a permissive environment by specifically expressing one or more essential genes only in the permissive environment, and/or expressing one or more lethal genes only in the non-permissive environment. Temporary viability in a non-permissive environment can be achieved by temporarily expressing one or more essential genes in a non-permissive environment, and/or temporarily delaying expression of one or more lethal genes in the non-permissive environment. Environmentally Limited Viability Systems are also disclosed involving coordinate expression of a combination of essential genes and lethal genes. Microorganisms containing an Environmentally Limited Viability System are useful for release into permissive and non-permissive environments. Temperature regulated Environmentally Limited Viability Systems and delayed death Environmentally Limited Viability Systems are particularly suited for delivery of expression products, such as antigens, using recombinant avirulent Salmonella by limiting their growth to the warmer environment inside the host, or by allowing growth for only a limited time in the host. Such microorganisms can be administered to protect humans or warm-blooded animals against bacterial, viral, mycotic and parasitic pathogens, especially those that colonize on or invade through mucosal surfaces. They can also be used for expression of gamete-specific antigens to induce immune responses to block fertilization, or to induce immune responses to tumor antigens.
Examples of Environmentally Limited Viability Systems are disclosed, including preferred forms involving viability limited by temperature and temporary viability in a non-permissive environment.